Altering inflammatory states of immune cells in vivo by modulating cellular activation states

This application is a U.S. National Phase Application based on International Pat. Application No. PCT/US2019/014209, filed Jan. 18, 2019, which claims the benefit of priority to U.S. Provisional Pat. Application No. 62/618,908, filed Jan. 18, 2018, both of which are incorporated by reference herein in their entirety.

The Sequence Listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is 2BD3077 ST25.txt. The text file is 145 KB, was created on Jun. 22, 2020, and is being submitted electronically via EFS-Web.

The current disclosure provides systems and methods to modulate the activation state of immune cells in vivo. The systems and methods can be used to transform immunosuppressive macrophages that support cancer growth and metastasis into highly activated tumoricidal macrophages.

A number of adverse physiological conditions are associated with immune system activation (e.g., autoimmune disorders) or immune system suppression (e.g., cancer). For example, macrophages are key immune effector cells that infiltrate cancerous tissue in high numbers. Within the tumor microenvironment, however, macrophages undergo a switch from an activated tumoricidal state to an immunosuppressive phenotype that actually facilitates tumor growth and metastasis. Pollard, Nat Rev Cancer 4, 71-78 (2004); Mantovani, et al., Nat Rev Clin Oncol (2017).

Understanding that immunosuppressed macrophages within the tumor microenvironment facilitate cancer growth and metastasis, much effort has been devoted to developing therapies that target immunosuppressive tumor-associated macrophages (TAMs). Many efforts to address TAMs have focused on killing the TAMs to alleviate immunosuppression in the tumor microenvironment. With this approach, however, the TAMs are simply replaced with newly-arriving macrophages at the tumor environment. Moreover, even when successful at killing some TAMs, most therapeutics developed to date have not been able to sufficiently penetrate into the tumor microenvironment. While some small molecule drugs and antibodies have shown some success, these approaches have suppressed all macrophages in the body, inducing dangerous side effects. Bowman & Joyce, Immunotherapy 6, 663-666 (2014). Thus, as is understood by everyone affected by cancer, more effective treatment strategies with fewer side effects are greatly needed.

The current disclosure provides systems and methods to modulate the function of immune cells in vivo. In particular embodiments, the systems and methods are used to reverse the immunosuppressive, tumor supporting state of tumor-associated macrophages (TAMs) and turn these TAMs into highly activated, tumor cell-killing macrophages. Thus, the systems and methods disclosed herein do not simply aim to kill TAMs, but instead redirects their activity from tumor-promoting to tumor-destroying. In particular embodiments, the systems and methods are used as a therapeutic to induce the killing of cancer cells and/or to reduce or prevent the growth or development of new cancer cells. Data disclosed herein shows that these systems and methods are able to completely eradicate and suppress ovarian cancer, a notoriously difficult cancer type to control.

The systems and methods disclosed herein can be used to alter the immunosuppressive state in a tumor, providing a mechanism to restructure the tumor microenvironment. In these embodiments, a restructured tumor microenvironment can render a tumor more susceptible to a companion treatment, such as a vaccine, a chimeric antigen receptor (CAR) therapy, and/or chemotherapy.

Importantly, the systems and methods disclosed herein can be used locally at the tumor microenvironment obviating the need to resort to systemic treatments that globally disrupt immune system homeostasis. Moreover, particular embodiments have been optimized to successfully infiltrate the tumor microenvironment.

Particular embodiments alter the activation states of immune cells in vivo by utilizing a particle to deliver nucleotides encoding activation regulators, such as transcription factors. A particularly useful particle has a positive core and a neutral or negatively-charged surface and delivers nucleotides encoding the transcription factor interferon-regulatory factor 5 (IRF5) in combination with the kinase IKKβ. A particle size of <130 nm ensures tumor infiltration. Moreover, the particles can include a TAM targeting ligand to direct more selective uptake of the particles by TAMs. As one example, TAMs express CD206, a cellular surface receptor that can be targeted by including mannose on the surface of the particles.

A number of adverse physiological conditions are associated with immune system activation (e.g., autoimmune disorders) or immune system suppression (e.g., cancer). For example, macrophages are key immune effector cells that infiltrate cancerous tissue in high numbers. However, within an immunosuppressive tumor milieu, they undergo a switch from an activated tumoricidal state to an immunosuppressive phenotype, which facilitates tumor growth and metastasis. These tumor-associated immunosuppressed macrophages (TAMs) are associated with poor prognosis (Komohara Y et al. (2014) Cancer science 105(1): 1-8). They induce angiogenesis, lymphogenesis, and stroma remodeling. They also play a key role in promoting tumor invasion and metastasis through secretion of the enzymes plasmin, uPA, matrix metalloproteinases (MMPs) and cathepsin B (Komohara, Y et al. (2016) Advanced drug delivery reviews 99: 180-185; Gocheva V et al. (2010) Genes Dev 24: 241-255; Wang R et al. (2011) Lung Cancer 74: 188-196). Apart from mediating tumor growth and progression, TAMs can also interact with other immune cells and suppress innate and adaptive antitumor immune responses.

Several small molecule drugs focus on blocking the localization of TAM-precursor cells to tumors by targeting the pathways involved in cell recruitment or expansion (i.e. inhibitors of the CSF-1/CSF-1R pathway (Pyon; teck et al. Nat Med 19, 1264-1272 (2013); Tap et al. N Engl J Med 373, 428-437 (2015)) or the CCL2 pathway (Nywening, et al. Lancet Oncol 17, 651-662 (2016)). These approaches require repeated systemic exposure to large doses of the small molecule drugs. Furthermore, clinical trials of these drugs showed low responses unless they were combined with cytoreductive therapies. Nywening, et al. Lancet Oncol 17, 651-662 (2016); Butowski et al. Neuro Oncol 18, 557-564 (2016). Furthermore, these small molecule approaches do not actively promote macrophage anti-tumor activity.

Conventional nanocarriers such as liposomes have been formulated with bisphosphonates or other antiproliferative agents to systemically destroy macrophages within a tumor (i.e. liposomal-clodronate) (Fritz et al., Front Immunol 5, 587 (2014)). Oncolytic viruses have also been used to deliver siRNA to silence immune-evasion pathways within tumors and indirectly promote phagocytosis of TAMs. (Chao et al., Curr Opin Immunol 24, 225-232 (2012)). The macrophages that are destroyed using these approaches, however, are naturally replaced by newly-arriving macrophages that similarly become immunosuppressive.

Antibodies have been developed to induce functional activation of TAMs. These approaches utilize antibodies to target defined antigen types within the tumor. Mantovani, et al., Nat Rev Clin Oncol (2017) Success of these antibodies, however, is limited by their low tumor penetration and heterogeneous distribution. Thurber et al., Adv Drug Deliv Rev 60, 1421-1434 (2008). They also do not address tumor escape variants that lack the antigen targeted by the antibody.

None of the described approaches directly and effectively reprogram TAMs to become activated tumoricidal macrophages, as disclosed herein. The systems and methods disclosed herein are significantly innovative because they allow the reprogramming of TAMs to become tumor-clearing macrophages while simultaneously reducing the tumor-promoting TAM burden. Currently, no other method exists that allow physicians to rationally reprogram TAMs for these therapeutic purposes. Mantovani, et al., Nat Rev Clin Oncol (2017); Gabrilovich & Nagaraj, Nat Rev Immunol 9, 162-174 (2009). This in and of itself can provide therapeutic benefit in the treatment of tumors. By modulating or restructuring the tumor microenvironment, the current disclosure also renders tumors more susceptible to other treatment types, such as vaccines, immunotherapies (e.g., CAR), and/or chemotherapies.

Particular embodiments utilize particles to provide cells with nucleotides encoding genes encoding activation regulators such as transcription factors (e.g., Interferon Regulatory Factors (IRFs)) and/or kinases (e.g., IKKβ). These activation regulators regulate macrophage polarization (). Macrophage polarization is a highly dynamic process through which the physiological activity of macrophages changes. As indicated, in most tumors, TAMs exhibit an immunosuppressed phenotype which can be an “M2” phenotype. By contrast, activated macrophages can exhibit an “M1” phenotype which results in tumor cell killing. Particular embodiments disclosed herein reverse the polarization of tumor-promoting TAMs into tumor-killing macrophages. This effect ameliorates the immunosuppressive milieu within the tumors by inducing inflammatory cytokines, activating other immune cells, and phagocytosing tumor cells.

“Macrophage activation” refers to the process of altering the phenotype or function of a macrophage from (i) an inactivated state to an activated state; (ii) a non-activated state to an activated state; (iii) an activated state to a more activated state; or (iv) an inactivated state to a non-activated state. An inactivated state means an immunosuppressed phenotype that facilitates tumor growth and metastasis. A non-activated state means that the macrophage neither facilitates tumor growth or metastasis nor promotes the killing of tumor cells. Activated means that the macrophage exhibits tumoricidal activity. In particular embodiments, the activated state results in an M1 phenotype as described more fully below. In particular embodiments, the inactivated state results in an M2 phenotype, also as described more fully below.

“Macrophage inactivation” refers to the process of altering the phenotype or function of a macrophage from (i) an activated state to a less activated state; (ii) an activated state to a non-activated state; (iii) an activated state to an inactivated state; or (iv) a non-activated state to an inactivated state. In particular embodiments, the inactivated state is M2. In particular embodiments, the activated state is M1.

In particular embodiments, one benefit of the disclosed systems and methods is that patients can be spared from systemic toxicities because inflammation induced by treatment remains localized at the treatment site. To achieve this benefit, locally infused particles target TAMs in the tumor milieu, (2) deliver nucleotides that selectively reprogram signaling pathways that control macrophage polarization, and (3) are completely degradable locally by physiological pathways (Sahin et al., Nat Rev Drug Discov 13, 759-780 (2014)).

Achieving high expression of exogenous nucleotides in solid tumors is challenging in vivo. Before the current disclosure, nucleotide delivery systems based on viruses or conventional nanocarriers such as liposomes were limited by their restricted diffusion within tumor tissue. Jain & Stylianopoulos, Nat Rev Clin Oncol 7, 653-664 (2010). To circumvent this barrier, particular embodiments utilize nanoparticles (NPs) with enhanced diffusivity so that the NPs deliver nucleotides to a large population of TAMs within a tumor. Particular embodiments utilize NPs <130 nm in size that carry a neutral surface charge. Particular embodiments can further include a targeting ligand attached to the surface of the NP. For example, di-mannose can be attached to the NP surface to enable more selective targeting to the mannose receptor (CD206) expressed on the TAM cell surface. Other TAM cell surface receptors that can be targeted include early growth response protein 2 (Egr2), CD163, CD23, interleukin (IL)27RA, CLEC4A, CD1a, CD1b, CD93, CD226, IL13-Ra1, IL-4r, IL-1R type II, decoy IL-1R type II, IL-10r, macrophage scavenging receptors A and B, Ym-1, Ym-2, Low density receptor-related protein 1 (LRP1), IL-6r, CXCR1/2, and PD-L1.

In particular embodiments, systems and methods disclosed herein include administering particles to a subject in need thereof. The particles are directed to macrophages present in tumors in the subject and are designed to be internalized by the macrophages. Once internalized, the particles further deliver one or more nucleotides having sequences that encode IRF5 and IKKβ. The one or more nucleotides modify the macrophages to express IRF5 and IKKβ. Without being bound by theory, the IKKβ kinase activates the IRF5 transcription factor by phosphorylation. Activated IRF5 then causes expression of type I interferon (IFN) genes, inflammatory cytokines, including tumor necrosis factor (TNF), IL-6, IL-12 and IL-23, and tumor suppressors. In M2 macrophages that have internalized one or more nucleotides encoding IRF5 and IKKβ, the expression of the aforementioned genes through IRF5 action leads to a phenotypic or functional switch of the macrophages from an M2 phenotype to an M1 phenotype, which enables the macrophages to kill or otherwise trigger the destruction of tumor cells, thereby treating cancer. In particular embodiments, the particles are internalized in the macrophages by phagocytosis. In particular embodiments, the particles are internalized in the macrophages by ligand-mediated endocytosis (e.g., CD-206-mediated endocytosis). In particular embodiments, delivery of the particles including the IRF5 and IKKβ genes into macrophages can include, e.g., (1) binding to the macrophages, (2) internalization of the particles by the macrophages, (3) escape from endocytic vesicles into the cytoplasm after internalization, (4) release of the one or more nucleotides, which (5) can be transported into the nucleus of the macrophages and (6) transcribed to deliver genes for expressing IRF5 and IKKβ.

Aspects of the disclosure are now described in more detail as follows: (1) macrophages and macrophage phenotypes; (2) cellular pathways to affect macrophage polarization; (3) nucleotides encoding activation regulators; (4) particles to deliver nucleotides; (5) targeting ligands to more selectively deliver nucleotides; (6) pharmaceutical compositions including particles; (7) methods of use; and (8) experimental examples.

(1) Macrophages and Macrophage Phenotypes. “Macrophage” refers to a white blood cell of the immune system differentiated from bone marrow derived monocytes. Macrophages are characterized by their phagocytic activity and their antigen presentation capacity. Macrophages are key players in both the innate and adaptive immune responses. Phenotypically macrophages express the surface marker F4/80 (Ly71) and may also express other surface markers such as CDIIb (Macl), CDIIc, CD14, CD40 or CD68.

Macrophages play an important role in both innate and adaptive immunity by activating T lymphocytes. In cancer, macrophages are one of the major populations of infiltrating leukocytes associated with solid tumors (Gordon S & Taylor P R (2005) Nature Reviews Immunology 5(12): 953-964). They can be recruited to the tumor site from surrounding tissues or by the tumor itself through the secretion of chemotactic molecules. Macrophages participate in immune responses to tumors in a polarized manner depending on their phenotype. “Phenotype” is used herein to refer to the physical attributes or biochemical characteristics of a cell as a result of the interaction of its genotype and the environment and can include functions of a cell.

Macrophages that activate Th1 T lymphocytes provide an inflammatory response and are often denoted as having an M1-polarized or “classically activated” phenotype. Macrophages in an activated state (i.e. M1 macrophages or macrophages having an M1 phenotype), also referred to as “killer macrophages,” inhibit cell proliferation, cause tissue damage, mediate resistance to pathogens, and possess strong tumoricidal activity. These macrophages can increase expression of mediators that are responsible for antigen presentation and costimulation; promoting infiltration of neutrophils to a tumor area leading to neutrophil-targeted tumor regression. An M1 phenotype can also be evidenced by increased antigen presentation as compared to a relevant control condition. In particular embodiments, an M1 phenotype can be evidenced by M1 macrophage production of reactive oxygen species (ROS) and nitric oxide (NO). NO has anti-proliferative effects integral for protection against pathogens and aberrant cells like tumor cells. In particular embodiments, an M1 phenotype can be evidenced by a pro-inflammatory state that induces Th1 immunity through the production of cytokines such as IL-12. In particular embodiments, macrophages in an activated state are classically activated macrophages that can phagocytose pathogens.

Beyond function, an M1 phenotype can also be evidenced by surface markers expressed by the macrophages; factors, proteins, or compounds produced by the macrophages upon polarization; or genes induced by the macrophages upon polarization. M1 polarization can lead to a phenotype evidenced by expression of CD80, CD86, iNOS, suppressor of cytokine signaling 3 (SOCS3), TNFα, IL-1, IL-6, IL-12, IL-23, Type I IFN, CXCL1, CXCL2, CXCL3, CXCL5, CXCL8, CXCL9, and CXCL10. In particular embodiments, an M1 phenotype includes an increase in expression of CD80. In particular embodiments, an M1 phenotype includes CD206−, MHCII+, CD11c−, and CD11b+.

On the other hand, macrophages that activate Th2 T lymphocytes provide an anti-inflammatory response and are often denoted as having an “M2” phenotype. Macrophages that are in an inactivated state (i.e. M2 macrophages or macrophages having an M2 phenotype), also referred to as “repair macrophages,” are involved in metazoan parasites containment, cell proliferation, tissue repair, tumor progression, anti-inflammation pathways, and immunosuppression. An M2 phenotype can reduce antigen presentation and decrease phagocytosis as compared to a relevant control condition. An M2 phenotype can also be evidenced by, for example, expression of one or more of arginase 1 (Arg1 (arginase activity is associated with pro-proliferative effects and tissue repair responses)), IL-10, TGF-β, PPArγ, KLF4, CD206 (MRC1), Dectin-1 (a signaling non-TLR pattern-recognition receptor), DC-SIGN (C-type lectin), scavenger receptor A, scavenger receptor B-1, CD163 (high affinity scavenger receptor for the hemoglobin-haptoglobin complex), chemokine receptors CCR2, CXCR1, and CXCR2, YM1 (chitinase 3-like 3), and Fizz1; and secretion of the chemokines CCL17, CCL22 and CCL24. In particular embodiments, macrophages in an inactivated state promote metastasis and/or resistance to chemotherapy. In particular embodiments, an M2 phenotype includes CD206+, MHCII−, CD11c+, and CD11b.

Table 1 provides particular combinations of criteria that can be used to distinguish an M1 phenotype from M2 phenotypes (including sub-phenotypes designated as M2a, M2b, M2c and M2d).

Assays to assess macrophage phenotype can take advantage of the different molecular signatures particular to the M1 or M2 phenotype. A commonly accepted marker profile for M1 macrophages is CD80+, whereas M2-macrophages can be characterized as CD163+. Thus, flow cytometry can be performed to assess for these markers. Driving macrophages towards a M1 type and away from a M2 type can also be assessed by measuring an increase of the IL-12/IL-10 ratio or the CD163−/CD163+ macrophage ratio. In particular embodiments, M1 versus M2 morphology can be assessed by light microscopy. In particular embodiments, phagocytosis assays may be used in conjunction with other assays to assess whether a macrophage is M1 type or M2 phenotype. Phagocytosis assays of different macrophage populations may be performed by incubating an entity to be phagocytosed with macrophages at a concentration that is consistent with their normalized total surface area per cell. The entity to be phagocytosed may be added to macrophage cultures. The entity to be phagocytosed may be, for example, labeled with a fluorescent label. Phagocytosis index may be determined by the median total fluorescence intensity measured per macrophage. Quantification of phagocytosis may be by, for example, flow cytometry. Tumor cell killing assays may also be utilized. In particular embodiments, an M1 phenotype includes reduced expression of signature M2 macrophage genes including SerpinB2 (inhibitor of urokinase-type plasminogen activator), CCL2 (C—C motif chemokine ligand 2), CCL11 (C—C motif chemokine ligand 11), and Retnla (resistin like alpha; Fizz1). In particular embodiments, an M1 phenotype includes increased expression of M1 differentiation genes including CCL5 (C—C motif chemokine ligand 5).

Gene expression (e.g., M1 expression of CD80, CD86 and/or other genes noted above) can be measured by assays well known to a skilled artisan. Methods to measure gene expression include NanoString nCounter® expression assays (NanoString Technologies, Inc., Seattle, WA), Northern blots, dot blots, microarrays, serial analysis of gene expression (SAGE), RNA-seq, and quantitative RT-PCR. Methods to measure gene expression products, e.g., protein level, include ELISA (enzyme linked immunosorbent assay), western blot, FACS, radioimmunological assay (RIA), sandwich assay, fluorescent in situ hybridization (FISH), immunohistological staining, immunoelectrophoresis, immunoprecipitation, and immunofluorescence using detection reagents such as an antibody or protein binding agents.

(2) Cellular Pathways to Affect Macrophage Polarization. Polarization of a macrophage towards an activated or inactivated phenotype results from macrophage interaction with a number of different molecules or environments. For example, M1 macrophage polarization is triggered by stimuli including Toll-like receptor (TLR) ligands (e.g. lipopolysaccharide (LPS), muramyl dipeptide, lipoteichoic acid, imiquimod, CpG), IFNα, TNFα, and macrophage colony-stimulating factor (GM-CSF). M2 polarized macrophages can be divided into subsets, depending on the stimuli that initiates the polarization: the M2a subtype is elicited by IL-4, IL-13 or fungal and helminth infections; M2b is elicited by IL-1 receptor ligands, immune complexes and LPS; M2c is elicited by IL-10, TGF-β and glucocorticoids; and M2d is elicited by IL-6 and adenosine. M2 macrophage polarization may also be triggered by IL-21, GM-CSF, complement components, and apoptotic cells. Macrophage polarization is also modulated by local microenvironmental conditions such as hypoxia.

The aforementioned molecules and environments affect macrophage polarization by triggering different intracellular signaling pathways involving transcription factors. Transcription factors that are involved in both M1 and M2 polarization include IRFs, signal transducers and activators of transcription (STAT), SOCS3 proteins, and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB). Mitogen-activated protein kinases (MAPK) also play a role in directing macrophage function towards either the M1 or M2 phenotype.

The IRF/STAT pathways, activated by such stimuli as IFNs and TLR signaling as discussed above, polarize macrophages to the M1 activation state via STAT1. On the other hand, such stimuli as IL-4 and IL-13 skew macrophages toward the M2 activation state via STAT6 (Sica A & Bronte V (2007) J Clin Invest 117: 1155-1166). These signaling events thus result in either the promotion of an inflammatory immune response and tumoricidal activity, as in the case of M1 macrophage polarization, or in the promotion of an immunosuppressive protumor response, as in the case of M2 macrophage polarization.

Some intracellular molecules implicated in the induction of an M1 phenotype include the G-protein coupled receptor, P2Y(2)R, which plays a role in inducing NO via NOS2 (Eun S Y et al. (2014) Int Immunopharmacol 18: 270-276); SOCS3, which activates NFκB/PI-3 kinase pathways to produce NO (Arnold C E et al. (2014) Immunology 141: 96-110); and growth and differentiation factor Activin A, which promotes M1 markers and down-regulates IL-10 (Sierra-Filardi E et al. (2011) Blood 117: 5092-5101).

Other intracellular molecules involved in induction of the M1 phenotype include IRFs. IRFs are a group of transcription factors with diverse roles, including virus-mediated activation of IFN, and modulation of cell growth, differentiation, apoptosis, and immune system activity. Members of the IRF family are characterized by a conserved N-terminal DNA-binding domain containing tryptophan (W) repeats.

IRF5 is a transcription factor that possesses a helix-turn-helix DNA-binding motif and mediates virus- and IFN-induced signaling pathways. It acts as a molecular switch that controls whether macrophages will promote or inhibit inflammation. IRF5 activates type I IFN genes, inflammatory cytokines, including TNF, IL-6, IL-12 and IL-23, and tumor suppressors as well as Th1 and Th17 responses. It is encoded by the human IRF5 gene located at chromosome 7q32 (OMIM ID 607218). It is appreciated that several isoforms/transcriptional variants of IRF5 exist. In particular embodiments, isoforms of human IRF5 include isoform 1 (UniProt Accession Q13568-1, SEQ ID NO: 1), isoform 2 (UniProt Accession Q13568-2, SEQ ID NO: 2), isoform 3 (UniProt Accession Q13568-3, SEQ ID NO: 3), isoform 4 (UniProt Accession Q13568-4, SEQ ID NO: 4), isoform 5 (UniProt Accession Q13568-5, SEQ ID NO: 5) and isoform 6 (UniProt Accession Q13568-6, SEQ ID NO: 6). In particular embodiments, isoforms of human IRF5 include isoform 1 encoded by a nucleotide sequence shown in SEQ ID NO: 23, isoform 2 encoded by a nucleotide sequence shown in SEQ ID NO: 24, isoform 3 encoded by a nucleotide sequence shown in SEQ ID NO: 25, isoform 4 encoded by a nucleotide sequence shown in SEQ ID NO: 26, isoform 5 encoded by a nucleotide sequence shown in SEQ ID NO: 27 and isoform 6 encoded by a nucleotide sequence shown in SEQ ID NO: 28. In particular embodiments, murine IRF5 includes an amino acid sequence shown in SEQ ID NO: 7. In particular embodiments, murine IRF5 is encoded by a nucleotide sequence shown in SEQ ID NO: 29. M1 macrophages have been shown to upregulate IRF5.

IRF1 and IRF8 also play critical roles in the development and function of myeloid cells, including activation of macrophages by proinflammatory signals such as IFN-γ. Dror N et al. (2007) Mol Immunol. 44(4):338-346. In particular embodiments, human IRF1 includes an amino acid sequence shown in SEQ ID NO: 8. In particular embodiments, human IRF1 is encoded by a nucleotide sequence shown in SEQ ID NO: 30. In particular embodiments, murine IRF1 includes an amino acid sequence shown in SEQ ID NO: 12. In particular embodiments, murine IRF1 is encoded by a nucleotide sequence shown in SEQ ID NO: 34. In particular embodiments, human IRF8 includes an amino acid sequence shown in SEQ ID NO: 11. In particular embodiments, human IRF8 is encoded by a nucleotide sequence shown in SEQ ID NO: 33. In particular embodiments, murine IRF8 includes an amino acid sequence shown in SEQ ID NO: 16. In particular embodiments, murine IRF8 is encoded by a nucleotide sequence shown in SEQ ID NO: 38.

IRF3 is a homolog of IRF1 and IRF2. It contains several functional domains including a NES, a DBD, a C-terminal IRF association domain and several regulatory phosphorylation sites. IRF3 is found in an inactive cytoplasmic form that upon serine/threonine phosphorylation forms a complex with CREB Binding Protein, a transcriptional coactivator. This complex translocates to the nucleus and activates the transcription of IFN-α and -β, as well as other interferon-induced genes. In particular embodiments, isoforms of human IRF3 include isoform 1 (UniProt Accession Q14653-1), isoform 2 (UniProt Accession Q14653-2), isoform 3 (UniProt Accession Q14653-3), isoform 4 (UniProt Accession Q14653-4), and isoform 5 (UniProt Accession Q14653-5). In particular embodiments, human IRF3 isoform 1 includes an amino acid sequence shown in SEQ ID NO: 9. In particular embodiments, human IRF3 isoform 1 is encoded by a nucleotide sequence shown in SEQ ID NO: 31. In particular embodiments, murine IRF3 includes an amino acid sequence shown in SEQ ID NO: 13. In particular embodiments, murine IRF3 is encoded by a nucleotide sequence shown in SEQ ID NO: 35.

IRF7 has been shown to play a role in the transcriptional activation of type I IFN genes. In particular embodiments, isoforms of human IRF7 include isoform A (UniProt Accession Q92985-1), isoform B (UniProt Accession Q92985-2), isoform C (UniProt Accession Q92985-3), and isoform D (UniProt Accession Q92985-4). In particular embodiments, human IRF7 isoform A includes an amino acid sequence shown in SEQ ID NO: 10. In particular embodiments, human IRF7 isoform A is encoded by a nucleotide sequence shown in SEQ ID NO: 32. In particular embodiments, murine IRF7 includes an amino acid sequence shown in SEQ ID NO: 14. In particular embodiments, murine IRF7 is encoded by a nucleotide sequence shown in SEQ ID NO: 36.

One or more IRF mutants that contribute to IRF activation may also be used. For example: phosphomimetic mutants of human variant 3/variant 4 of IRF5 (isoform 4, SEQ ID NO: 4) that substitute amino acid residues S425, S427, S430, S436 with residues mimicking phosphorylation, such as aspartic acid residues (Chen W et al. (2008) Nat Struct Mol Biol. 15(11): 1213-1220); phosphomimetic mutants of human variant 5 of IRF5 (isoform 2, SEQ ID NO: 2) that substitute amino acid residues T10, S158, S309, S317, S451, and/or S462 with residues mimicking phosphorylation, such as aspartic acid residues (Chang Foreman H-C et al. infra); mutation of human IRF5 isoform a (variant 1, isoform 3, SEQ ID NO: 3) and isoform b (variant 2, isoform 1, SEQ ID NO: 1) residues S156, S158 and T160 to residues mimicking phosphorylation, such as aspartic acid residues, for constitutive nuclear accumulation of IRF5 (Lin R et al. (2005) J Biol Chem 280(4): 3088-3095); and IRF3 phosphomimetic mutants that substitute amino acid residue S396 of IRF3 with residues mimicking phosphorylation, such as aspartic acid (Chen W et al. infra). In particular embodiments, a fusion protein of murine IRF7/IRF3 includes Asp (D) mutations at four serine and one threonine residues in the IRF3 association domains (SEQ ID NO: 15), conferring constitutive activation and translocation of the fusion protein (Lin R et al. (1998) supra; Lin et al. (2000) Molecular and Cellular Biology 20: 6342-6353). In particular embodiments, a fusion protein of murine IRF7/IRF3 including D mutations at four serine and one threonine residues in the IRF3 association domains is encoded by a nucleotide sequence shown in SEQ ID NO: 37. In particular embodiments, a murine IRF8 mutant includes substitution of Lysine (K) at amino acid residue 310 with Arginine (R) (SEQ ID NO: 17). In particular embodiments, a murine IRF8 mutant including a substitution of K at amino acid residue 310 with R is encoded by a nucleotide sequence shown in SEQ ID NO: 39. Small ubiquitin-like modifiers (SUMO) bound to IRF8 primarily at K310 inhibit activation of IRF8 responsive genes. Sentrin-specific protease 1 (SENP1) targets SUMO 2/3. The activity of SENP1 “deSUMOylates” IRF8 (and other proteins) and causes IRF8 to go from a repressor of M1 macrophage differentiation to an activator (directly and through transactivation activities). Preventing SUMO binding to IRF8 by mutation of the K310 residue increases IRF8 specific gene transcription 2-5 fold (see Chang T-H et al. (2012) supra).

Particular embodiments of the present disclosure include engineered IRF transcription factors. In particular embodiments, engineered IRF transcription factors include IRFs that lack a functioning autoinhibitory domain and are therefore insensitive to feedback inactivation (Thompson et al. (2018) Front Immunol 9: 2622). For example, a human IRF5 with 2-3-fold increase in activity can be obtained by deleting aa 489-539 of the human IRF5 protein (Barnes et al. (2002) Mol Cell Biol 22: 5721-5740). In particular embodiments, an autoinhibitory domain of IRF4, a transcription factor that promotes an M2 phenotype, can be deleted or mutated to generate a more active IRF4 in the context of treating an autoimmune disease. In particular embodiments, an autoinhibitory domain of an IRF is found at the carboxy terminus of the IRF protein. In particular embodiments, engineered IRF transcription factors include IRFs that lack one or more functioning nuclear export signals (NES) to entrap IRFs in the nucleus and therefore enhance transcription. For example, nuclear accumulation of human IRF5 can be achieved by mutating the NES of human IRF5 by replacing two leucine residues with alanine (L157A/L159A) (Lin et al. (2000) Molecular and Cellular Biology 20: 6342-6353). In particular embodiments, engineered IRF transcription factors include fusions of one or more IRFs, fusions of fragments of one or more IRFs, and fusions of mutated IRFs.

NFκB is also a key transcription factor related to macrophage M1 activation. NFκB regulates the expression of a large number of inflammatory genes including TNFα, IL1B, cyclooxygenase 2 (COX-2), IL-6, and IL12p40. NFκB activity is modulated via the activation of the inhibitor of kappa B kinase (IKK) trimeric complex (two kinases, IKKα, IKKβ, and a regulatory protein, IKKγ). When upstream signals converge at the IKK complex, they first activate IKKβ kinase via phosphorylation, and activated IKKβ further phosphorylates the inhibitory molecule, inhibitor of kappa B (I-κB). This results in the proteosomal degradation of I-κB and the release of NFκB p65/p50 heterodimer from the NFκB/I-κB complex. The NFκB p65/p50 heterodimer is then translocated to the nucleus and binds to the promoters of inflammatory genes.

IKKβ is an activating kinase for NFκB as well as other transcription factors such as IRF5. IKKβ similarly phosphorylates several other signaling pathway components including FOXO3, NCOA3, BCL10, IRS1, NEMO/IKBKG, NFκB subunits RELA and NFKB1, as well as the IKK-related kinases TBK1 and IKBKE. In particular embodiments, isoforms of human IKKβ include isoform 1 (UniProt Accession O14920-1, SEQ ID NO: 18), isoform 2 (UniProt Accession O14920-2 SEQ ID NO: 19), isoform 3 (UniProt Accession O14920-3 SEQ ID NO: 20), and isoform 4 (UniProt Accession O14920-4 SEQ ID NO: 21). In particular embodiments, isoforms of human IKKβ include isoform 1 encoded by a nucleotide sequence shown in SEQ ID NO: 40, isoform 2 encoded by a nucleotide sequence shown in SEQ ID NO: 41, isoform 3 encoded by a nucleotide sequence shown in SEQ ID NO: 42, and isoform 4 encoded by a nucleotide sequence shown in SEQ ID NO: 43. In particular embodiments, murine IKKβ includes an amino acid sequence shown in SEQ ID NO: 22. In particular embodiments, murine IKKβ is encoded by a nucleotide sequence shown in SEQ ID NO: 44.

The present disclosure provides for the co-expression of IRF transcription factors with one or more molecules that can activate the IRFs to effect TAM reprogramming to an activated state for tumor killing. In particular embodiments, co-expression strategies include: co-expression of IRF5 and IKKβ; co-expression of IRF5 and TANK-binding kinase-1 (TBK-1), TNF receptor-associated factor 6 (TRAF6) adaptor, receptor interacting protein 2 (RIP2) kinase, and/or NFκB kinase-ε (IKKε) (Chang Foreman H-C et al. (2012) PLoS One 7(3): e33098); co-expression of IRF5 and protein kinase DNA-PK (Ryzhakov G et al. (2015) J of Interferon & Cytokine Res 35(2): 71-78); co-expression of IRF5 and protein kinase tyrosine kinase BCR-ABL (Massimo M et al. (2014) Carcinogenesis 35(5):1132-1143); and co-expression of IRF5 or IRF8 with one or more components of the COP9 signalosome (Korczeniewska J et al. (2013) Mol Cell Biol 33(6):1124-1138; Cohen H et al. (2000) J Biol Chem 275(50):39081-39089).

In particular embodiments, the teachings of the current disclosure can be applied in the management of conditions triggered by hyper-immune activation (e.g., autoimmune diseases). Macrophages play key roles in autoimmune diseases such as systemic lupus erythematosus, multiple sclerosis, rheumatoid arthritis, and Sjögren's syndrome (Ushio et al. World J Immunol 2017; 7(1): 1-8). Thus, cellular pathways that support an immunosuppressive M2 phenotype are also described.

An activation regulator implicated in the induction of the M2 phenotype is Krüppel-like factor 4 (KLF-4). KLF-4 coordinates with STAT6 to induce M2 genes such as Arg-1, CD206 (Mrc1, mannose receptor), Fizz1 (resistin-like α) and peroxisome proliferator-activated receptor gamma (PPArγ), and to inhibit M1 genes such as TNFα, Cox-2, CCL5 and iNOS. The nuclear receptor, PPARγ, has been shown to regulate genes involved in oxidative metabolism and activation of the M2 phenotype (Odegaard J I et al. (2007) Nature 447: 1116-1120).

The cytokine IL-21 mediates M2 polarization by decreasing NOS2 expression and increasing STAT3 phosphorylation (Li S N et al. (2013) Mediators Inflamm 2013, 548073).

IRF4 negatively regulates TLR signaling in a MyD88 independent manner to drive the M2 phenotype (Satoh T et al. (2010) Nat Immunol 11, 936-944). In particular embodiments, human IRF4 is UniProt Accession Q15306. BMP-7 also induces M2 polarization in vitro via activation of the SMAD-PI3K-Akt-mTOR pathway (Rocher C et al. (2013) Plos One 8: e84009).

Transcription factor glucorticoid-induced leucine zipper (GILZ). GILZ is a dexamethasone-inducible gene that mediates glucocorticoid (GC) actions in a variety of cell types and it can induce the suppressive M2 macrophage phenotype. GILZ expression is rapidly and ubiquitously induced by GCs, and the protein product interacts with known transcription factors, such as NF-κB, Raf-1, TORC2, AP-1, Ras, and C/EBPs, inhibiting the expression of pro-inflammatory genes. Thus, GILZ could mimic the therapeutic anti-inflammatory effects of GCs while avoiding the detrimental ones (Ronchetti, S. et al. Front Endocrinol (Lausanne) 2015; 6: 170). In particular embodiments, GILZ is human GILZ of amino acid sequence shown in SEQ ID NO: 110. In particular embodiments, GILZ is human GILZ encoded by a nucleotide sequence shown in SEQ ID NO: 111.

As indicated, hypoxia also influences macrophage polarization through hypoxia inducible factors HIF-1α and HIF-2α. HIF-1α regulates NOS2 expression and supports emergence of an M1 phenotype while HIF-2α regulates Arg1 expression and supports emergence of an M2 phenotype (Takeda N et al. (2010) Genes Dev 24: 491-501).